Project Summary/Abstract Viruses with positive-sense RNA (+RNA) genome compose a large group of plant and animal viruses, and many human viruses of medical concerns belong to this group of viruses. All known +RNA viruses form and replicate within vacuole-like structures in the cytoplasm, called replication complex (RC). Viral RC is made by viruses through reorganization of cellular organelle membranes, and it provides a favorable microenvironment for the viruses to replicate. Nevertheless, it has been obscure whether and how the host immune system counteracts such viral RCs. Understanding the host immune defense strategy against viral RC may allow us to develop broadly applicable antiviral strategies against +RNA viruses. We recently found that interferon-gamma (IFNG) inhibits the replication of murine norovirus (MNV) at the stage of RC formation. Intriguingly, this antiviral activity of IFNG depends on a protein complex involved in cellular autophagy. Autophagy is an evolutionarily conserved pathway that sequesters cytoplasmic materials in double-membrane-bound autophagosomes and delivers them to the lysosome for degradation. To form a globular autophagosome, the microtubule-associated-protein-1-light- chain-3 (LC3) conjugation system is essential. We found that only the LC3 conjugation system of autophagy, but not the lysosomal degradation through autophagy, is required for IFNG to inhibit MNV RC formation. Interestingly, IFNG also requires the same LC3 conjugation system, but not the lysosomal degradation, to disrupt a cytosolic vacuole containing a protist parasite Toxoplasma gondii. Through a comparative mechanism study of MNV and T. gondii models, we found that the LC3 conjugation system was required to recruit IFN-inducible GTPases, immunity related GTPases (IRGs) and guanylate binding proteins (GBPs), to the RC of MNV. Both IRGs and GBPs are known to be targeted to the membrane of vacuoles containing bacterium, protist, or fungus. The targeted membranes are vesiculated and eventually the vacuoles rupture, leading to the death of exposed pathogens. Similarly, the GTPases were required for IFNG to disrupt MNV RCs and consequently to inhibit the replication of MNV in both mouse and human systems. This is a novel and paradigm-shifting antiviral mechanism of IFNG, indicating a common effector mechanism against disparate pathogens replicating in cytosolic membranous shelters, including +RNA virus as well as bacterium, protist, and fungus. Our long-term goal is to harness the medical benefits based on the functional mechanism of this antiviral immune defense against viral RCs. The overall objective of this proposal, as the next step to pursue that goal, is to determine how the RC of MNV is detected and disrupted by the immune system. Our central hypothesis is that MNV RC is detected by the LC3 conjugation system of the autophagy pathway and then the structure/function of RC is disrupted by the IFN-inducible GTPases recruited via the LC3 conjugation system. The new fundamental knowledge created in this study will have significant positive impact on human health because it will provide a novel insight into antiviral mechanisms used by interferons and potentially new therapeutic targets of intervention for viral diseases.